Allogeneic hepatocyte transplantation without immunosuppression - Project Summary Hepatocyte transplantation has the potential to replace orthotopic liver transplantation for many liver diseases, especially genetic metabolic disorders. Cell therapy has significant advantages over liver directed gene therapy, including durability, the ability to administer repeat doses, and its applicability to multiple disorders, representing “one drug for multiple diseases”. However, significant obstacles to more wide-spread use of liver cell therapy remain. First, hepatocyte engraftment is rate limiting and it is difficult to achieve the replacement index needed for clinical benefit. Second, hepatocytes from organ donors are an allogeneic cell source and require immune suppression to persist. Here, we propose to overcome these barriers by using genetic enhancement of donor hepatocytes, making them immune stealthy and enabling selective growth expansion after engraftment. Three specific aims are designed to systematically explore multiple approaches to donor cell enhancement. In Aim 1, we will render donor hepatocytes resistant to acetaminophen (APAP) by knockout of Cypor. Two models of genetic liver disease (alkaptonuria and gyrate atrophy) will be used to test the efficacy of donor hepatocyte selection with APAP. In Aim 2, immune stealthy hepatocytes will be generated by knockout of class I HLA genes followed by class II HLA genes under inflammatory conditions. Gene-edited hepatocytes will be tested in allotransplantation models. In Aim 3, we will combine the manipulations to render donor hepatocytes immune stealthy and expandable. Fully enhanced allogeneic donor hepatocytes will be tested in a phenylketonuria disease mouse model. Together these experiments will lead to proof-of-principal that immune stealthy, expandable hepatocytes can overcome the major obstacles of therapeutic hepatocyte transplantation.
